Loss of TSC1 or TSC2 drives lineage-infidelity and hamartoma formation in a renal organoid model of angiomyolipoma Renal angiomyolipomas (R-AMLs) are hamartomatous kidney growths which stain positively for markers of adipocytic, vascular, smooth muscle, and melanocytic lineages. These lesions possess loss of function mutations in either TSC1 or TSC2, which are canonical negative regulators of mTORC1 signalling. To date, there exists no in vitro or in vivo model which faithfully recapitulates the architectural and molecular complexity of R-AMLs. Considering these lesions can be detected very early in life- even congenitally- we hypothesized they arise as a consequence of aberrant tissue development. To test this hypothesis, we generated TSC1-/- and TSC2-/- mutants in four human pluripotent stem cell (hPSC) backgrounds using CRISPR/Cas9 genome engineering. Wild type hPSCs differentiated into renal organoids express markers of the glomerulus, proximal and distal tubules, in a topology that resembles human nephron patterning. Remarkably, wild type renal organoids downregulate mTORC1 signalling compared to adjacent undifferentiated cells. In contrast, both TSC1-/- and TSC2-/- hPSCs exhibit substantial lineage infidelity upon differentiation, staining positive for adipocytic and melanocytic markers which are absent in matched wild type controls. Additionally, knockout lines formed nodular growths with disorganized architecture, resembling the hamartomatous organization of R-AML lesions. These lesions exhibit hyperactive mTORC1 signalling, consistent with human pathology. Together, these data suggest three primary findings: loss of TSC1/2 drives lineage infidelity; TSC1/2 may be required for architectural organization of the kidney parenchyma; and a developmental approach to R-AML modelling may best recapitulate the human disease.